Factor VII is a polypeptide involved in the blood clotting process. Today, Factor VIIa can be made by recombinant techniques (rFVIIa) and is widely used as a pro-haemostatic agent. Factor VII (human wild-type) has been described in U.S. Pat. No. 4,784,950. rFVIIa offers today a rapid and highly effective pro-haemostatic response in haemophilic individuals experiencing bleeding. Advantageously, rFVIIa can be used for treating haemophilic individuals that cannot be treated with other coagulation factor products due to antibody formation. Also individuals suffering from Factor VII deficiency or individuals having a normal coagulation system but still experiencing excessive bleeding can be treated successfully with rFVIIa.
Several parameters need to be considered when developing a medicament comprising a polypeptide such as, e.g., Factor VIIa. By example, the medicament needs to be effective, safe and lead to good patient compliance. Moreover, the medicament may be formulated for parenteral administration using pharmaceutically acceptable excipients, which will have to meet with the approval of various world-wide medical regulatory agencies. For the purpose of parenteral administration, it is highly desirable that the formulation is approximately isotonic and that the pH of the formulation is in a physiologically suitable range upon injection/infusion, otherwise it may result in pain and discomfort for the patient. For a general review of protein formulations, see, for example, Cleland et al.: The development of stable protein formulations: A closer look at protein aggregation, deamidation and oxidation, Critical Reviews in Therapeutic Drug Carrier Systems 1993, 10(4): 307-377; and Wang et al., Parenteral formulations of proteins and peptides: Stability and stabilizers, Journal of Parenteral Science and Technology 1988 (Supplement), 42 (2S).
However, for medicaments comprising polypeptides the safety may directly be related to the physical and chemical stability of the polypeptide. Being a polypeptide, Factor VII or a Factor VII-related polypeptide is susceptible to physical degradation, including denaturation and aggregation such as the formation of soluble or insoluble aggregates in the form of dimers, oligomers and polymers, or to chemical degradation, including for example, hydrolysis, deamidation and oxidation. Consequently, the said physical and chemical instability may lead to loss of activity of the Factor VII polypeptide, formation of toxic and immunogenic degradation products, serious risk of introducing thrombosis upon injection of the degraded Factor VII polypeptides, clogging of needles used for injections and risk of non-homogeneity, to name just a few.
Thus, it is essential to provide compositions comprising Factor VII polypeptides that is stabilised against physical and chemical degradation.
Today, recombinantly-made FVII polypeptide is provided as freeze-dried product that is meant to be stored at temperatures between about 2 and about 8° C. The requirement of cooled conditions causes a burden to and is inconvenient for the manufacturer or provider as well as the end user (the patient).
The actual recombinantly-made FVII product is NovoSeven® (Novo Nordisk A/S, Denmark) that consists of 1.2 mg recombinant human Factor VIIa, 5.84 mg NaCl, 2.94 mg CaCl2, 2H2O, 2.64 mg Glycylglycine, 0.14 mg polysorbate 80 and 60.0 mg mannitol. When reconstituted by 2.0 ml of water for injection (WFI), the pH is 5.5 and the thus prepared FVII-containing solution is sufficiently stable for 24 hours at room temperature.
The present investigators have found that upon storage of the lyophilised NovoSeven® product for 6 months at 25° C. about 6 to 7% w/w of the initial content of the rFVIIa is present in the form of aggregates.
Thus, compositions comprising Factor VII polypeptides need to be stabilised so as allowing storage and handling at ambient temperatures. However, the instability of polypeptides relates to several parameters and it is impossible to predict the proper manner of stabilising a Factor VIIa or a Factor VII-related polypeptide.
One approach of stabilising a protein relates to removal of water from the protein, e.g. such as providing the protein in the form of a lyophilised cake, the final matter obtained in a freeze-drying process. However, the freeze-drying process itself is also harmful to proteins; during freeze-drying, the protein solution is first cooled until adequately frozen and bulk water in the protein solution will form ice at this stage. The protein is hereby prone to freeze-induced stress resulting in deformation and precipitation. In the next step, the so-called primary drying stage, the ice sublimes and in the secondary drying stage, adsorbed or bound water is removed under elevated temperatures. During this water removal, the proteins may loose their proper conformation that is provided mainly through hydrogen bonding.
Therefore, to preserve protein conformation, activity and stability during freeze-drying, the polypeptide solution needs to be supplemented with sufficient amounts of proper excipients with cryoprotectant and/or lyoprotectant properties so as to protect the protein from freeze-induced stress and/or stress during removal of water, respectively.
Additionally, when providing a lyophilised product, an essential feature relates to the properties of the lyophilised cake. It needs to have good properties as to its form and structure, i.e. it should not collapse in that such collapsed cakes can be hard or even impossible to dissolve (reconstitute) before use. Conversely, the physical structure of the lyophilised cake may not be too loosen and soft. Therefore, one or more so-called bulking agents are added to the protein solution before freeze-drying.
Other publications of interest regarding stabilisation of polypeptides are as follows:
U.S. Pat. No. 2,001,0031721 A1 (American Home Products) concerns highly concentrated, lyophilised, and liquid Factor IX formulations.
WO 97/26909 (Genetics Institute) concerns lyophilised preparations of Factor IX suitable for storage and administration. The preparations may comprise sucrose or mannitol as a cryoprotectant.
WO 95/28954 (Genetics Institute) concerns preparations of Factor IX suitable for storage and administration. The preparations may comprise sucrose as a cryoprotectant.
It is an objective of the present invention to provide stable compositions of Factor VII polypeptides, substantially without the presence of degradation products and without decreased activity of the Factor VII polypeptides, preferable after prolonged storage at ambient conditions, e.g. for at least 6 months. Furthermore, it is an objective that the stable compositions are suitable for parenteral administration so as not to cause any inconvenience for the patient.